Five-Azacytidine inhibits bacterial DNA (cytosine-5) methylases when bacteria are grown in its presence. This inhibition is irreversible and is specific for cytosine methylases; adenine methylases are unaffected. We have found that inhibition can be reproduced in vitro if DNA containing 5-azacytosine is used as the inhibitor. To be an effective inhibitor the DNA must be double-stranded. Time-dependent inhibition can be demonstrated if the enzyme is preincubated with the inhibitor and the rate of inhibition is decreased if substrate DNA is present in the preincubation. Inhibitory activity is lost if the DNA is digested with the cognate restriction enzyme but not when digested with a noncognate restriction enzyme. The purpose of this proposal is to determine the mechanism of this site-specific irreversible inhibition of the cytosine DNA methylases. Radioactive labeled azacytosine-containing DNA will be prepared and its interaction with the purified EcoRII methylase studied. If irreversible binding of the DNA to the methylase occurs, the sequences bound to the enzyme will be determined. The effect of azacytidine on DNA methylation in vivo will also be determined. Using bromouracil and methyl-labeled methionine, we shall determine if methylcytosine can be formed in cells containing azacytosine in their DNA.